XU, Q; LI, S; DUAN, C: Differential subcellular localization of IGFBP-4 and IGFBP-5 is determined by their distinct C-domains

IGFBP-4 and -5 are closely related members of the IGFBP gene family. Thy share similar domain structures, substantial sequence identity, and the ability to bind IGFs with high affinities. Despite these similarities, they have differential subcellular localization and different biological actions. In smooth muscle cells (SMCs), IGFBP-5, but not -4, is found in the nucleus. IGFBP-5 potentiates IGF-stimulated cell proliferation and migration, whereas IGFBP-4 acts as an inhibitor. To understand the underlying structural basis, six IGFBP chimera (N5L5C4, N5L4C4, N4L5C4, N4L4C5, N5L4C5, and N4L5C5) were generated by exchanging the N, L, and C domain between IGFBP-4 and �5. These six chimera, as well as wild type IGFBP-4 and -5, were tagged with EGFP and transfected to cells. IGFBP-5/EGFP was seen exclusively in the nuclei, whereas IGFBP-4/EGFP was observed only in the cytoplasm. All the chimera lacking the IGFBP-5 C-domain exhibited little or no nuclear presence. Replacement of the IGFBP-4 C-domain with that of IGFBP-5 yielded three chimeric IGFBPs and they were predominantly nuclear. These results suggest that the determinants specifying the differential subcellular localization of IGFBP-4 and -5 are localized in their C-domains. To further determine the critical motifs, several IGFBP-5 point mutants were generated by replacing charged residues in the C-domain. Mutation of K217/R218 into 217A/218A reduced the nuclear presence of IGFBP-5 by >70%. Replacing charged residues between 201 to 208 also resulted in significant decreases in nuclear localization, albeit to a less degree. Double mutation of these two motifs caused a further decrease. These data suggest that these two clusters of charged residues in the C-domain of IGFBP-5 are important for nuclear localization of IGFBP-5 (Supported by NIHRO1HL60679).